As mitochondria are central hubs for energy metabolism and formation of intermediate metabolites, they must be able to transmit signals that communicate alterations in its functioning to the nucleus so that cells can remodel metabolism in response. Mitochondrial-nuclear signaling is particularly important when mitochondrial function is compromised by environmental toxicants, including heavy metals. However, the nature and dynamics of these inter-organelle signals and the mechanisms by which they are transduced are poorly understood. Given the importance of heme-regulated transcription factors that control diverse cellular processes from energy metabolism to the anti-oxidant stress response to cell proliferation, and the exquisite sensitivity of mitochondrial heme homeostasis to heavy metals, we propose that mitochondrial- derived heme signals the cellular response to heavy metal toxicity through mitochondrial-nuclear retrograde regulation. However, the chemical and genetic tools available to study the transmission of heme-based signals do not exist. In the current grant application, the focus is to generate and apply a genetically encoded ratiometric fluorescent heme sensors in cell and animal models to study the transduction of mitochondrial-derived intra- and inter-organ heme signals in response to heavy metal toxicity. During the R21 phase, the intent is develop and apply the first ratiometric heme sensors for quantitative heme imaging in yeast and mammalian cell models of heavy metal toxicity. Finally, the goal is to (a) characterize the thermodynamics of heme binding to the heme sensors and apply them site-specifically in the yeast mitochondrial matrix, inter-membrane space, cytosol, and nucleus to quantitatively image heme signals in response to lead toxicity; and (b) apply sensors in a compartment specific manner in mammalian cell models of lead toxicity. Successful completion of these aims will provide the impetus to begin the R33 phase in which studies will be expanded to develop heme sensors that can be utilized for simultaneous imaging of heme between cellular compartments, and that are oxidation state-specific, and apply them to animal models of Pb toxicity. In this phase the focus is to (a) diversify the color palate of the heme sensors for simultaneous fluorescence imaging between subcellular compartments and develop Fe3+ and Fe2+ heme specific sensors; and (b) deploy these sensors in mammalian cell lines and a C. elegans model of environmental toxicity. Altogether, these studies will result in the first sensors for quantitative imaging of labile heme relevant to its role in cell signaling and establish heme a a vital mitochondria-derived signaling molecule that initiates the adaptation to heavy metal toxicity in both cell and animal models.